Surgical pathology is a field of medicine concerned with the evaluation of tissue removed from patients during surgery to address medical conditions. Many surgical procedures require that tissue removed during surgery is microscopically evaluated for the presence of pathologies which may require additional therapy or surgery. Most microscopically evaluated tissue is preserved with formaldehyde or other fixative, embedded into paraffin or similar media, and then cut into optically thin sections which are stained or otherwise labeled. The evaluation is most commonly performed on a transillumination microscope where an image is generated by absorption of light transmitted through the specimen. This method of evaluation is lengthy because of the extensive chemical processing required in order to cut and stain the tissue and may delay surgical procedures to such an extent that it is costly or even impractical in some scenarios.
Because the lengthy processing precludes histological evaluation during many types of surgery, patients undergoing the resection of cancer or other pathology, including cancer of the breast, may require multiple surgeries to achieve complete removal of pathological tissue. For example, in breast conserving surgery for carcinoma of the breast, a majority of patients may require a second surgery to achieve complete treatment of the disease due to the finding of microscopic areas of carcinoma present on the surface of the excised tissue following the conclusion of surgery. Intraoperative imaging techniques such as Mohs micrographic surgery (MMS) can reduce the rate of second surgeries by freezing tissue, cutting it into thin sections, and then staining and evaluating sections on a conventional transillumination pathology microscope to determine the adequacy of resection. These techniques are time-consuming and of insufficient sensitivity to be applicable in the treatment of many cancers, including those of the breast. Alternatively, standard paraffin-embedded histopathology (PEH) is both cost effective and highly sensitive, but is too time consuming (nearly 1 day processing time for fixation and paraffin embedding) to be used in many surgical scenarios. Therefore a need exists for alternative devices and methods of evaluating tissue for the presence of pathology.
Procedures such as MMS and PEH incur long processing times primarily because of the need to physically section tissue into thin (typically on the order of 5 micron) slices which can be stained, mounted on slides and imaged on a transillumination pathology microscope. The sectioning process is necessary because transillumination microscopy relies on transmitting light through a specimen where it is absorbed by dyes producing an image by attenuating certain colors relative to others. The resulting color image represents a single image plane cut within a larger tissue specimen, and is used to render a diagnosis by inspection of tissue or individual cells for signs of pathology (for example, enlarged or irregularly shaped cells or cell nuclei) by a trained pathologist. Commonly this image is generated from the H&E stain, which is composed of hematoxylin, a dye that primarily stains the nuclei of cells purple, and eosin, a counterstaining dye that renders many other tissue components, such as cytoplasm, stroma, and collagen, pink. For this process to work, tissue must be cut thin enough that light can transmit through the specimen, effectively forming a single image plane by physical cutting (sectioning) of the plane from the larger specimen. In the case of cancer of the breast, a diagnosis is rendered primarily based on the appearance, orientation and density of cell nuclei.
To reduce sample preparation time, optical depth sectioning was proposed in the past, in which advanced microscopy techniques are used to selectively image a single 2D plane within a larger, intact 3D specimen. These techniques avoid the lengthy physical sectioning process associated with PEH and MMS by selectively imaging a single plane using optical methods and so can be used in scenarios where minimization of imaging time is important. Among devices and methods, optically sectioned reflectance confocal imaging of large specimens combined with low magnification imaging for guidance has been proposed, primarily for applications such as skin cancer. However, reflectance confocal alone cannot provide the molecular contrast required to image cell nuclei, and therefore cannot be used to render a diagnosis in many surgical pathology applications, including breast conserving surgery where diagnostic criteria depend critically on examining the location, organization and appearance of cell nuclei. Other methods have been proposed based on techniques such as full field optical coherence tomography, but these too lack the ability to resolve nuclei.
Most microscopy techniques that can generate an optically sectioned image do not produce an image through optical absorption of transmitted light and so do not intrinsically produce an image resembling conventional transillumination microscopy. Therefore, interpretation of images produced by these methods is difficult or ambiguous for the vast majority of pathologists and surgeons trained in conventional transillumination microscopy. Instead of transillumination, most optically sectioning techniques operate in epi-illumination mode, where illumination and imaging both occur from the same surface of a specimen. Furthermore, most produce images based on the total power of light reflected by a specimen (reflectance confocal microscopy or optical coherence tomography) or a spectral shift in the wavelength of light returned from a specimen relative to the wavelength of illumination (e.g. fluorescence, second harmonic generation or Raman scattering). In either case, an image is produced that is brighter when substance of interest is present, and darker otherwise. However, it has been demonstrated that it is possible to produce virtual transillumination images, which can precisely reproduce the diagnostic features present in conventional transillumination microscopy, from tissue using computational methods. These methods, called virtual transillumination microscopy (VTM) enable pathologists trained in existing pathology techniques to perform diagnoses more rapidly by producing images that depict nuclei and other cellular components as they would appear in a transillumination microscope with an H&E slide. However, the integration of VTM methods into devices and methods for surgical pathology imaging has received less attention, and so is not commonly available for use in surgical procedures.
A further problem concerns the large scale of many surgical excisions and the limited time to evaluate them during a surgery. As microscopic evaluation requires high magnification imaging with limited field of view, locating areas of pathology on large surgical samples can be time consuming. For example, a typical 20× magnification image used for confocal imaging may cover less than one square millimeter, whereas a typical breast excision may have a surface area of more than 10,000 square millimeters. However, surgical time is costly, and a maximum amount of time during which a patient can be reasonably kept in surgery exists, which makes comprehensive imaging of the entirety of very large excisions impractical. Unfortunately, many previously proposed devices and methods of surgical imaging depend on being able to comprehensively image the entire surface at high resolution, stitch together a mosaic of many individual images, and then use the mosaic image for diagnosis, a process which is impractically slow for large specimens.
Presently used methods for surgical pathology typically employ various markings such a sutures or colored inks that a placed on a specimen during or after excision but before histological examination to guide the evaluation of histology. These markings are often used both for orienting the tissue with respect to the surgical cavity from which it was extracted, and to indicate the position of histologically relevant aspects of tissue. For example, in cancer of the breast treated with breast conserving surgery, excised tissue is removed from the patient, inked with up to 6 different colors, each indicating a different aspect (side) of the specimen. Following inking, the specimen is dissected into a number of thin slices with inked aspects on edges indicating the histologically relevant surgical margins. Although the dissection process results in the loss of the original shape of the tissue specimen, by referencing color inks present on the edges of the slices, a trained pathologist can locate the original surface of the tissue and assess the proximity of any pathology to the margin or edge of the excision. If pathology is present on or too close to the edge of the excision, the surgical margin can be deemed insufficient and an additional surgical resection required, and the location of the excision guided based on the color of the ink. Inking procedures are also used in other surgeries, including many treatments for dermatological malignancies such as basal cell carcinoma. Consequently, it is essential that imaging systems used for evaluating surgical pathology incorporate methods and devices for assessing the location of sutures, surgical inks, or other exogenous markings. However, imaging sutures, surgical inks, or other exogenous markings is difficult or impossible under fluorescence imaging, reflectance confocal imaging or optical coherence tomography, necessitating other methods such as white light imaging or narrow band imaging that can be used to guide the user, in real-time, diagnostically relevant regions while avoiding imaging areas that are not relevant.